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Rad51 paralog complex Rad55–Rad57 acts as a molecular chaperone during homologous recombination. Roy et al.

Upasana Roy1
12 Feb 2020-bioRxiv (Mendeley Data)-Vol. 1
TL;DR: Single–molecule imaging is used to reveal that the Saccharomyces cerevisiae Rad51 paralog complex Rad55–Rad57 promotes the assembly of Rad51 recombinase filaments through transient interactions, providing evidence that it acts as a classical molecular chaperone.
Abstract: Summary Homologous recombination (HR) is essential for the maintenance of genome integrity. Rad51 paralogs fulfill a conserved, but undefined role in HR, and their mutations are associated with increased cancer risk in humans. Here, we use single–molecule imaging to reveal that the Saccharomyces cerevisiae Rad51 paralog complex Rad55–Rad57 promotes the assembly of Rad51 recombinase filaments through transient interactions, providing evidence that it acts as a classical molecular chaperone. Srs2 is an ATP–dependent anti–recombinase that downregulates HR by actively dismantling Rad51 filaments. Contrary to the current model, we find that Rad55– Rad57 does not physically block the movement of Srs2. Instead, Rad55–Rad57 promotes rapid re– assembly of Rad51 filaments after their disruption by Srs2. Our findings support a model in which Rad51 is in flux between free and ssDNA–bound states, the rate of which is dynamically controlled though the opposing actions of Rad55–Rad57 and Srs2.
Citations
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Journal ArticleDOI
TL;DR: In this paper , the replication fork block stimulates a unique recombination pathway dependent on Rad51 strand invasion and Rad52-Rad59 strand annealing activities, Mph1/Rad5 fork remodelers, Mre11/Exo1/Dna2 resection machineries, Rad1-Rad10 nuclease and DNA polymerase δ.
Abstract: Replication stress and abundant repetitive sequences have emerged as primary conditions underlying genomic instability in eukaryotes. To gain insight into the mechanism of recombination between repeated sequences in the context of replication stress, we used a prokaryotic Tus/Ter barrier designed to induce transient replication fork stalling near inverted repeats in the budding yeast genome. Our study reveals that the replication fork block stimulates a unique recombination pathway dependent on Rad51 strand invasion and Rad52-Rad59 strand annealing activities, Mph1/Rad5 fork remodelers, Mre11/Exo1/Dna2 resection machineries, Rad1-Rad10 nuclease and DNA polymerase δ. Furthermore, we show recombination at stalled replication forks is limited by the Srs2 helicase and Mus81-Mms4/Yen1 nucleases. Physical analysis of the replication-associated recombinants revealed that half are associated with an inversion of sequence between the repeats. Based on our extensive genetic characterization, we propose a model for recombination of closely linked repeats that can robustly generate chromosome rearrangements.

17 citations

Journal ArticleDOI
TL;DR: In this paper , the replication fork block stimulates a unique recombination pathway dependent on Rad51 strand invasion and Rad52-Rad59 strand annealing activities, Mph1/Rad5 fork remodelers, Mre11/Exo1/Dna2 resection machineries, Rad1-Rad10 nuclease and DNA polymerase δ.
Abstract: Replication stress and abundant repetitive sequences have emerged as primary conditions underlying genomic instability in eukaryotes. To gain insight into the mechanism of recombination between repeated sequences in the context of replication stress, we used a prokaryotic Tus/Ter barrier designed to induce transient replication fork stalling near inverted repeats in the budding yeast genome. Our study reveals that the replication fork block stimulates a unique recombination pathway dependent on Rad51 strand invasion and Rad52-Rad59 strand annealing activities, Mph1/Rad5 fork remodelers, Mre11/Exo1/Dna2 resection machineries, Rad1-Rad10 nuclease and DNA polymerase δ. Furthermore, we show recombination at stalled replication forks is limited by the Srs2 helicase and Mus81-Mms4/Yen1 nucleases. Physical analysis of the replication-associated recombinants revealed that half are associated with an inversion of sequence between the repeats. Based on our extensive genetic characterization, we propose a model for recombination of closely linked repeats that can robustly generate chromosome rearrangements.

16 citations

Journal ArticleDOI
TL;DR: It is built upon previous data to show that SMARCAL1, ZRANB3 and HLTF have unequal biochemical capacities, explaining why they have non-redundant functions, and provides a possible mechanism explaining previous cellular experiments implicating RAD51 and BCDX2 in fork reversal.
Abstract: Abstract SMARCAL1, ZRANB3 and HLTF are required for the remodeling of replication forks upon stress to promote genome stability. RAD51, along with the RAD51 paralog complex, were also found to have recombination-independent functions in fork reversal, yet the underlying mechanisms remained unclear. Using reconstituted reactions, we build upon previous data to show that SMARCAL1, ZRANB3 and HLTF have unequal biochemical capacities, explaining why they have non-redundant functions. SMARCAL1 uniquely anneals RPA-coated ssDNA, which depends on its direct interaction with RPA, but not on ATP. SMARCAL1, along with ZRANB3, but not HLTF efficiently employ ATPase driven translocase activity to rezip RPA-covered bubbled DNA, which was proposed to mimic elements of fork reversal. In contrast, ZRANB3 and HLTF but not SMARCAL1 are efficient in branch migration that occurs downstream in fork remodeling. We also show that low concentrations of RAD51 and the RAD51 paralog complex, RAD51B–RAD51C–RAD51D–XRCC2 (BCDX2), directly stimulate the motor-driven activities of SMARCAL1 and ZRANB3 but not HLTF, and the interplay is underpinned by physical interactions. Our data provide a possible mechanism explaining previous cellular experiments implicating RAD51 and BCDX2 in fork reversal.

7 citations

Journal ArticleDOI
TL;DR: In this article , the authors summarize the current understanding of the meiotic functions of RAD51 and DMC1 and the role of their positive and negative modulators during plant meiosis.
Abstract: Homologous recombination during meiosis is crucial for the DNA double-strand breaks (DSBs) repair that promotes the balanced segregation of homologous chromosomes and enhances genetic variation. In most eukaryotes, two recombinases RAD51 and DMC1 form nucleoprotein filaments on single-stranded DNA generated at DSB sites and play a central role in the meiotic DSB repair and genome stability. These nucleoprotein filaments perform homology search and DNA strand exchange to initiate repair using homologous template-directed sequences located elsewhere in the genome. Multiple factors can regulate the assembly, stability, and disassembly of RAD51 and DMC1 nucleoprotein filaments. In this review, we summarize the current understanding of the meiotic functions of RAD51 and DMC1 and the role of their positive and negative modulators. We discuss the current models and regulators of homology searches and strand exchange conserved during plant meiosis. Manipulation of these repair factors during plant meiosis also holds a great potential to accelerate plant breeding for crop improvements and productivity.

5 citations

Journal ArticleDOI
TL;DR: In this article , the BRCA2 C-terminal RAD51-binding segment (TR2) acts in a dominant manner to overcome the effect of BRC4.

4 citations

References
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Book ChapterDOI
TL;DR: The yeast Saccharomyces cerevisiae is now recognized as a model system representing a simple eukaryote whose genome can be easily manipulated and made particularly accessible to gene cloning and genetic engineering techniques.
Abstract: Publisher Summary The yeast Saccharomyces cerevisiae is now recognized as a model system representing a simple eukaryote whose genome can be easily manipulated. Yeast has only a slightly greater genetic complexity than bacteria and shares many of the technical advantages that permitted rapid progress in the molecular genetics of prokaryotes and their viruses. Some of the properties that make yeast particularly suitable for biological studies include rapid growth, dispersed cells, the ease of replica plating and mutant isolation, a well-defined genetic system, and most important, a highly versatile DNA transformation system. Being nonpathogenic, yeast can be handled with little precautions. Large quantities of normal baker's yeast are commercially available and can provide a cheap source for biochemical studies. The development of DNA transformation has made yeast particularly accessible to gene cloning and genetic engineering techniques. Structural genes corresponding to virtually any genetic trait can be identified by complementation from plasmid libraries. Plasmids can be introduced into yeast cells either as replicating molecules or by integration into the genome. In contrast to most other organisms, integrative recombination of transforming DNA in yeast proceeds exclusively via homologous recombination. Cloned yeast sequences, accompanied by foreign sequences on plasmids, can therefore be directed at will to specific locations in the genome.

3,547 citations

Journal ArticleDOI
TL;DR: The discovery of ghrelin indicates that the release of GH from the pituitary might be regulated not only by hypothalamic GH-releasing hormone, but also by gh Relin derived from the stomach, which plays important roles for maintaining GH release and energy homeostasis in vertebrates.
Abstract: Small synthetic molecules called growth hormone secretagogues (GHSs) stimulate the release of growth hormone (GH) from the pituitary. They act through the GHS-R, a G protein-coupled receptor whose ligand has only been discovered recently. Using a reverse pharmacology paradigm with a stable cell line expressing GHS-R, we purified an endogenous ligand for GHS-R from rat stomach and named it "ghrelin," after a word root ("ghre") in Proto-Indo-European languages meaning "grow." Ghrelin is a peptide hormone in which the third amino acid, usually a serine but in some species a threonine, is modified by a fatty acid; this modification is essential for ghrelin's activity. The discovery of ghrelin indicates that the release of GH from the pituitary might be regulated not only by hypothalamic GH-releasing hormone, but also by ghrelin derived from the stomach. In addition, ghrelin stimulates appetite by acting on the hypothalamic arcuate nucleus, a region known to control food intake. Ghrelin is orexigenic; it is secreted from the stomach and circulates in the bloodstream under fasting conditions, indicating that it transmits a hunger signal from the periphery to the central nervous system. Taking into account all these activities, ghrelin plays important roles for maintaining GH release and energy homeostasis in vertebrates.

2,740 citations

Journal ArticleDOI
24 Feb 1989-Cell
TL;DR: Northern analysis of strains containing plasmid inserts with various promoter mutations suggests that the stimulation in recombination is mediated by events initiating within the integrated plasmID sequences.

1,641 citations

Journal ArticleDOI
TL;DR: HR accessory factors that facilitate other stages of the Rad51- and Dmc1-catalyzed homologous DNA pairing and strand exchange reaction have also been identified.
Abstract: Homologous recombination (HR) serves to eliminate deleterious lesions, such as double-stranded breaks and interstrand crosslinks, from chromosomes. HR is also critical for the preservation of repli- cation forks, for telomere maintenance, and chromosome segrega- tion in meiosis I. As such, HR is indispensable for the maintenance of genome integrity and the avoidance of cancers in humans. The HR reaction is mediated by a conserved class of enzymes termed recombinases. Two recombinases, Rad51 and Dmc1, catalyze the pairing and shuffling of homologous DNA sequences in eukaryotic cells via a filamentous intermediate on ssDNA called the presynaptic filament. The assembly of the presynaptic filament is a rate-limiting process that is enhanced by recombination mediators, such as the breast tumor suppressor BRCA2. HR accessory factors that facil- itate other stages of the Rad51- and Dmc1-catalyzed homologous DNA pairing and strand exchange reaction have also been identified. Recent progress on elucidating the mechanisms of action of Rad51 and Dmc1 and their cohorts of ancillary factors is reviewed here.

1,542 citations

Journal ArticleDOI
17 Sep 2004-Cell
TL;DR: The cellular response to DSBs and DNA replication stress is likely directed by the Mre11 complex detecting and processing DNA ends in conjunction with Sae2 and by RP-A recognizing single-stranded DNA and recruiting additional checkpoint and repair proteins.

938 citations


"Rad51 paralog complex Rad55–Rad57 a..." refers background or result in this paper

  • ...GFP–tagged Rad55–Rad57 forms DNA repair foci in vivo in response to IR (Lisby et al., 2004)....

    [...]

  • ...Under these conditions, there was extensive co–localization of Rad55–Rad57 and Rad51 on the ssDNA (Figure 2B), providing a satisfying explanation for the observation that Rad51 is required for Rad55–57 foci formation in vivo (Lisby et al., 2004)....

    [...]

  • ...The finding that Rad55–Rad57 did not bind to RPA–ssDNA was consistent with the observation that Rad55–Rad57 foci do not form in vivo without Rad51 (Lisby et al., 2004)....

    [...]

  • ..., 2017); note, use of fluorescent RPA is necessary because fluorescent Rad51 fusion proteins are not fully functional (Lisby et al., 2004; Waterman et al., 2019)....

    [...]